Recent developments in fluorescence microscopy geared for single-molecule detection, optical sectioning and three-dimensional data acquisition, used in combination with object-identification algorithms, now allow sufficient temporal and spatial resolution to visualize the sequential recruitment and intracellular traffic of proteins, lipids, and pathogens in living cells including viruses and bacteria. The Kirchhausen laboratory has a longstanding interest in understanding the mechanisms underlying entry of toxins, viruses and bacteria into cells, and how these processes relate to the organization and intracellular traffic of vesiculo-tubular membrane carriers. As part of this effort, we have devoted substantial efforts to developing an imaging suite containing state-of-the-art microscopes and supporting software suited for data collection and analysis with high spatial and temporal resolution. It is also a priority to maintain an imaging suite that is 100% accessible to all users, so that after initial training, any investigator can perform their work with total flexibility and independence. In this proposal we outline plans for a NERCE Imaging Core facility that will provide access to contemporary tools and expertise for live-cell and single molecule imaging aimed towards, but not limited to, quantitative descriptions of mechanisms of invasion for bacterial and viral pathogens into mammalian cells, of viral replication, and of molecular aspects related to toxin entry into cells.